WO2018023208A1 - Method for preparing nicotinamide mononucleotide - Google Patents

Abstract

Disclosed is a method for preparing nicotinamide mononucleotide. Raw materials nicotinamide, pyrophosphoric acid or a salt thereof and AMP react under the catalytic effects of nicotinamide phosphoribosyltransferase and adenine phosphoribosyltransferase so as to obtain nicotinamide mononucleotide.

Description

 Method for preparing nicotinamide mononucleotide

 Technical field

 [0001] The present invention relates to the field of molecular biology and biotechnology, and more particularly to a method for preparing a nicotinamide mononucleotide using a biocatalytic technique.

 Background technique

 [0002] Nicotinamide mononucleotide (NMN) is a biochemical substance present in biological cells, which is transformed into a biological cell after being adenylated by nicotinamide nucleotide adenosyltransferase. The important substance that depends on the survival of nicotinamide adenine dinucleotide (NAD, also known as coenzyme I), and homologous directly involved in adenosine transfer in vivo, its level in biological cells directly affects the concentration of NAD, in biological cells It plays an important role in energy generation and is harmless to the human body.

[0003] Up to now, it has been found that nicotinamide mononucleotides have many health care uses such as delaying aging, treating senile diseases such as Parkinson's, regulating insulin secretion, affecting mRNA expression, and the like, and more applications are being continuously developed. . With the increasing awareness of the medicinal and health effects of nicotinamide mononucleotides and the widespread use of chemicals as a reaction substrate, the demand for nicotinamide mononucleotides is increasing.

 [0004] At present, the preparation method of NMN mainly includes the following three types: 1. yeast fermentation method; 2. chemical synthesis method; 3. biocatalysis method. Among them, the chemical synthesis method has the disadvantages of high cost and the production of chiral compounds; and the NMN produced by the yeast fermentation method contains certain organic solvent residues; the biocatalytic method does not contain the solvent residue, and there is no chiral problem and is prepared. NMN is the same as the same type in the body and has become the most green and environmentally friendly NMN preparation method. The existing biocatalytic method for preparing NMN is generally based on nicotinamide and 5'-phosphoribosyl-1'-pyrophosphate (PRPP), and Nicotinamide e phosphoribosyltransferase (abbreviated to Nampt). Preparation of NMN under catalysis. However, due to the high market price and limited source of PRPP, the production cost of the biocatalytic method is high, which seriously restricts its application and development.

[0005] Therefore, it is necessary to develop a new method for preparing NMN using biocatalysis technology without using PRPP as a substrate. technical problem

 [0006] In view of the problems in the preparation method of the existing nicotinamide mononucleotide mentioned in the above background art, an object of the present invention is to provide a novel method for preparing NMN by using a biocatalytic technique, which is avoidable PRUse high-priced and limited-source PRPP as a substrate, which has the advantages of low price, environmental protection, pollution-free, and suitable for large-scale industrial production.

 Problem solution

 Technical solution

 [0007] In order to test the above object, the inventors have finally developed a method for preparing a nicotinamide mononucleotide after a long period of extensive experimentation, which is characterized in that: nicotinamide, pyrophosphoric acid or a salt thereof and AMP are used as raw materials. The reaction is carried out under the catalysis of nicotinamide phosphoribosyltransferase and adenine phosphoribosyltransferase to obtain a nicotinamide mononucleotide.

 [0008] According to the international system nomenclature of enzymes, the EC numbers of the enzymes used in the above methods are: nicotinamide phosphoribosyltransferase EC 2.4.2.12, adenine phosphoribosyltransferase EC 2.4.2.7.

 [0009] The specific forms of the various enzymes used in the above methods include an enzyme solution, an enzyme lyophilized powder, an enzyme-containing cell, and various immobilized enzymes and immobilized enzyme-containing cells, which may be in the form of unpurified crude enzyme. It may also be in a partially purified or fully purified form.

 [0010] In order to improve the stability and recyclability of the enzyme to be used, in order to better accomplish the above catalytic reaction and further reduce the cost, it is preferred to use an immobilized enzyme in the above method. The immobilized enzyme is prepared by: diluting the enzyme to a protein content of 5-10 mg/ml with a washing enzyme buffer (0.02 M Tris-HCl/0.001 M EDTA, pH 7.0 solution), and then diluting the enzyme with PB solution (2.0 mol / L potassium dihydrogen phosphate, pH 7.5) was mixed in equal volume, then added to the enzyme immobilization carrier (50 mg enzyme / gram carrier), reacted at 25 ° C in a shaker (rotation speed 150 rpm) 20 small Inches. After the reaction is completed, it is filtered with a filter bag and washed with a washing enzyme buffer for 5-6 times to obtain an immobilized enzyme. Among the enzyme-immobilized carriers, epoxy type LX-3000, silica, activated carbon, glass beads, and macroporous poly-N-aminoethyl acrylamide-polyethylene can be used.

[0011] Preferably, the pyrophosphate salt is a sodium salt of pyrophosphate, including sodium pyrophosphate, disodium pyrophosphate.

Preferably, the reaction is carried out at a temperature of 30 to 50 ° C and a pH of 6.5 to 8.5.

More preferably, the reaction has the highest conversion of hydrazine at a temperature of 35-45 ° C and a pH of 7.0-8.0. Preferably, the reaction is carried out in the presence of Mg 2+ and K + .

Preferably, the reaction is carried out in Tris-HCl buffer.

 [0016] Preferably, the concentration of the nicotinamide is 1-150 mM, and the concentration of the pyrophosphate or its salt is 1-100 mM

The concentration of the AMP is 1-100 mM.

[0017] Preferably, the molar ratio of nicotinamide, pyrophosphoric acid or its salt and AMP in the raw material is 1-4:1-2:1, and the raw material can be sufficiently reacted according to the ratio of the raw materials, and the conversion thereof is carried out. The rate is 80<3⁄4-100<3⁄4. Since the price of AMP is the highest among the three raw materials, this ratio can greatly reduce the production cost. More preferably, the molar ratio of nicotinamide, pyrophosphoric acid or its salt to AMP in the raw material is 2:1.5:1, the conversion calculated by the substrate AMP is 100%, and the cost is the lowest.

[0018] The nicotinamide mononucleotide crude product solution obtained after the completion of the reaction can be subjected to filtration, purification and drying treatment by a conventional technique known in the art, that is, a nicotinamide single nucleotide product can be obtained.

Preferably, the nicotinamide phosphoribosyltransferase used in the above method is a protein of the following (a) or (b):

 (a) a protein having the amino acid sequence of SEQ ID NO: 3,

 (b) having one or more amino acids substituted, deleted or added in the (a) defined amino acid sequence and having a nicotinamide and PRPP as a substrate having a higher amino acid sequence than the parent represented by SEQ ID NO: 2. The nicotinamide phosphoribosyltransferase catalytically active protein derived from).

 The above-described nicotinamide phosphoribosyltransferase is a site-directed mutagenesis of the gene of the parent nicotinamide phosphoribosyltransferase derived from the nucleotide sequence of Meiothermus ruber DSM 1279, as shown in SEQ ID NO: 1, after PCR amplification. A series of highly catalytically active nicotinamide phosphoribosyltransferase mutants were obtained by inserting an appropriate vector and subsequently screening on LB+ kanamycin medium. The high catalytic activity of these mutants can greatly reduce industrial application organisms. The cost of catalytic technology for the production of nicotinamide mononucleotides has high industrial application value.

 Preferably, the nicotinamide phosphoribosyltransferase has at least one mutation selected from at least one of the following positions compared to the amino acid sequence set forth in SEQ ID NO: 2: position 180, position 182 , 231rd, 298th, 338th and 377th.

More preferably, the nicotinamide phosphoribosyltransferase has at least one of the following mutations: F180A, F180 W, A182Y, E231A, E231Q, D298A, D298N, D298E, D338N, D338E, D37 7A, D377N and D377E.

 Advantageous effects of the invention

 Beneficial effect

 [0025] 1. Compared with the preparation method of the existing nicotinamide mononucleotide, the method provided by the invention overcomes the defects of the chemical synthesis method and the yeast fermentation method, and successfully avoids the price and the source limitation. The use of PRPP, the conversion rate calculated by the substrate AMP is as high as 100%, and belongs to the most environmentally friendly and pollution-free preparation method of the current nicotinamide mononucleotide, which is suitable for large-scale industrial production and low in price.

 2. The nicotinamide phosphoribosyltransferase used in the method provided by the present invention is a mutant obtained by artificially induced site-directed mutagenesis, and the enzyme activity of the mutant is greatly improved compared with the existing wild type. The enzymatic activity assay uses nicotinamide and PRPP as substrates. The enzyme has a catalytic activity of 1.2-6.9 times that of the parent. Such high catalytic activity allows it to be used as a crude enzyme without purification, or only a part of it. Purification, which makes the cost of catalyzing the production of NMN by using the nicotinamide phosphoribosyltransferase mutant provided by the present invention greatly reduced, has high market competitiveness, and can meet the demand for applying the biocatalytic method of NMN to large-scale industrial production. .

Embodiments of the invention

 The present invention will be further described in detail below with reference to the specific embodiments. The following examples are illustrative of the invention. The invention is not limited to the following examples, and the specific conditions are not indicated in the examples, according to conventional conditions or manufacturing. The conditions suggested by the business are carried out.

 [0028] The specific implementation process of the method for preparing nicotinamide mononucleotide provided by the present invention is as follows:

[0029] The raw materials are prepared by dissolving each raw material in water, and the composition of the substrate solution is 1-150 mM of nicotinamide, 1-100 mM pyrophosphoric acid or a salt thereof, 1-100 mM AMP, and 1--30 mM MgCl ₂ , l-20 mM KCl and 50-100 mM Tris-HCl buffer, adjust the pH to 6.5-8.5. The following catalytic enzymes were then added to the substrate solution: nicotinamide phosphoribosyltransferase 1-100 g/L substrate solution, adenine phosphoribosyltransferase 1-100 g/L substrate solution. After stirring uniformly, the reaction was carried out, stirring was continued during the reaction (stirring speed 50 rpm), the reaction temperature was controlled to 30-50 ° C, and the pH was maintained at 6.5-8.5. After 1-8 hours of reaction, a crude nicotinamide mononucleotide solution is obtained, which is filtered, purified and dried to obtain a finished nicotinamide single nucleotide.

[0030] The enzyme used in the following examples, except for the nicotinamide phosphoribosyltransferase mutant, was from Meiother The nucleotide sequence of mus ruber DSM 1279 is obtained by artificially induced site-directed mutagenesis of the parent nicotinamide phosphoribosyltransferase shown in SEQ ID NO: 1, and the remaining nicotinamide phosphoribosyltransferase and adenine phosphoribosyltransferase are both It is an enzyme lyophilized powder purchased directly from the market.

Embodiment 1

Preparation of Nicotinamide Mononucleotide

[0033] The substrate solution was added to the reaction vessel, containing 1 mM nicotinamide, 1 mM disodium pyrophosphate, 1 mM AM P, 1 mM MgCl ₂ , 1 mM KC1, and 50 mM Tris-HCl buffer, and adjusted to pH. 6.5-7.0. The following catalytic enzymes were then added: nicotinamide phosphoribosyltransferase lg/L substrate solution, adenine phosphoribosyltransferase lg/L substrate solution. After stirring uniformly, the reaction was carried out, stirring was continued during the reaction (stirring speed 50 rp m), the reaction temperature was controlled at 30 ° C, and the pH was maintained at 6.5-7.0. After 1 h of reaction, a crude nicotinamide mononucleotide solution (containing NMN 0.9 mM) was obtained, which was filtered, purified and dried to obtain a finished nicotinamide single nucleotide.

 Example 2

 Preparation of Nicotinamide Mononucleotide

[0036] The substrate solution was added to the reaction vessel, containing 40 mM nicotinamide, 20 mM pyrophosphoric acid, 20 mM AMP, 10 mM MgCl ₂ , 7 mM KC1, and 70 mM Tris-HCl buffer, and adjusted to pH 7.0-7.5. . Then, the following catalytic enzymes were added: nicotinamide phosphoribosyltransferase 30 g/L substrate solution, adenine phosphoribosyltransferase 30 g/L substrate solution. After the mixture was stirred well, the reaction was continued, stirring was continued during the reaction (stirring speed 5 O rpm), the reaction temperature was controlled at 35 ° C, and the pH was maintained at 7.0-7.5. After 3 hours of reaction, a crude nicotinamide mononucleotide solution (containing NMN18.9 mM) was obtained, which was filtered, purified and dried to obtain a finished nicotinamide single nucleotide.

 Example 3

 Preparation of Nicotinamide Mononucleotide

[0039] A substrate solution was added to the reaction vessel containing 100 mM nicotinamide, 70 mM sodium pyrophosphate, 70 mM A MP, 20 mM MgCl ₂

15 mM KC1 and 100 mM Tris-HCl buffer were adjusted to pH 7.5-8.0. Then, the following catalytic enzymes were added: nicotinamide phosphoribosyltransferase 65 g/L substrate solution, adenine phosphoribosyltransferase 65 g/L substrate solution. After stirring evenly, the reaction was carried out, and stirring was continued during the reaction (stirring speed 50 rpm), and control was carried out. The reaction temperature was 40 ° C and the pH was maintained at 7.5-8.0. After 5 hours of reaction, a crude nicotinamide mononucleotide solution (containing NMN 65.7 mM) was obtained, which was filtered, purified and dried to obtain a finished nicotinamide single nucleotide.

 Embodiment 4

 Preparation of Nicotinamide Mononucleotide

[0042] A substrate solution was added to the reaction vessel containing 150 mM of nicotinamide, 100 mM sodium pyrophosphate, 100 mM AMP, 30 mM MgCl ₂

 20 mM KC1 and 100 mM Tris-HCl buffer were adjusted to pH 8.0-8.5. Then, the following catalytic enzymes were added: nicotinamide phosphoribosyltransferase 100 g/L substrate solution, adenine phosphoribosyltransferase 100 g/L substrate solution. After stirring well, the reaction was carried out, stirring was continued during the reaction (stirring speed 50 rpm), the reaction temperature was controlled at 50 ° C, and the pH was maintained at 8.0-8.5. After 8 h of reaction, a crude nicotinamide mononucleotide solution (containing NMN 96.5 mM) was obtained, which was filtered, purified and dried to obtain a finished nicotinamide single nucleotide.

 Embodiment 5

 Preparation of Nicotinamide Mononucleotide

Preparation of immobilized enzyme: Dilute nicotinamide phosphoribosyltransferase and adenine phosphoribosyltransferase to a protein content of 5, using a washing enzyme buffer (0.02 M Tris-HCl/0.001 M EDTA, pH 7.0 solution) -10m g/ml, and then mix the enzyme dilution with the PB solution (2.0mol/L potassium dihydrogen phosphate, pH 7.5) in an equal volume, and then add the enzyme immobilized carrier epoxy type LX-3000 (50 mg enzyme / gram Carrier), reacted at 25 ° C for 20 hours in a shaker (rotation speed 150 _Φ m). After completion of the reaction, the mixture was filtered through a filter bag and washed with a washing enzyme buffer for 5-6 times to obtain immobilized nicotinamide phosphoribosyltransferase and immobilized adenine phosphoribosyltransferase, respectively.

[0046] A substrate solution was added to the reaction vessel containing 75 mM nicotinamide, 75 mM disodium pyrophosphate, 50 mM AMP, 15 mM MgCl ₂

 10 mM KC1 and 100 mM Tris-HCl buffer were adjusted to pH 7.0-7.5. Then, the following catalytic enzymes were added: immobilized nicotinamide phosphoribosyltransferase 50 g/L substrate solution, immobilized adenine phosphoribosyltransferase 50 g/L substrate solution. After the mixture was stirred well, the reaction was continued, stirring was continued during the reaction (stirring speed 5 O rpm), the reaction temperature was controlled at 37 ° C, and the pH was maintained at 7.0-8.0. After 5 hours of reaction, a crude nicotinamide mononucleotide solution (containing NMN 48.8 mM) was obtained, which was filtered, purified and dried to obtain a finished nicotinamide mononucleotide.

Example 6 Preparation of Nicotinamide Phosphoribosyltransferase Mutant

 The preparation process of the artificially induced site-directed mutagenesis nicotinamide phosphoribosyltransferase used in the method provided by the present invention is roughly as follows: First, a vector plasmid containing a parent nicotinamide phosphoribosyltransferase gene is constructed, and then a site-directed mutagenesis is set. The site and the amino acid species after mutation, and then synthesize appropriate primers, and use the vector plasmid containing the parent nicotinamide phosphoribosyltransferase gene as a template to PCR-amplify the DNA fragment, assemble the amplified DNA fragment, and fully expand the PCR amplification. Mutant gene. The full-length mutant gene is then cloned into an appropriate vector and transformed into an appropriate host cell, and a positive clone having nicotinamide phosphoribosyltransferase activity is selected by culture. Finally, the plasmid DNA is extracted from the positive clone, and the DNA sequence analysis is performed to determine the introduced mutation. After the target fragment is inserted into the vector, the LB+ kanamycin medium is selected for screening, thereby obtaining a series of high catalytic activity. Nicotinamide phosphoribosyltransferase mutant.

 In the above preparation method, any suitable vector may be employed, for example, it may be a prokaryotic expression vector such as pRSET and pES21, etc.; and may be a cloning vector such as pUC18/19 and pBluscript-SK. In the present invention, pRSET-A is preferably used as a vector, and the host cell of the vector may be a prokaryotic cell including Escherichia coli, or a eukaryotic cell including Saccharomyces cerevisiae and P. pastoris.

 [0051] 1. Construction of a vector plasmid containing a parent nicotinamide phosphoribosyltransferase gene

 The gene sequence of the parent nicotinamide phosphoribosyltransferase from Meiothermus ruber DSM 1279 published by the gene bank (GenBank accession number: CP001743.1) was subjected to full sequence synthesis (completed by a commercial synthesis company). The synthesized product was digested with restriction endonucleases Ndel and BamHI and digested with the same restriction endonucleases Ndel and BamHI, pRSET-A (from Invitrogen,

USA) ligation, the plasmid pRSET- _n ampt. The nucleotide sequence of the cloned parent nicotinamide phosphoribosyltransferase was determined by DNA sequencing as shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO: 2.

 [0053] Preparation of Nicotinamide Phosphoribosyltransferase Mutant

[0054] The PCR amplification reaction system is: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgS0 ₄ , 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng DNA template, and 400 nM upstream primer and 400 nM downstream primer, adjusted to 50 μm with sterile water Rise.

[0055] The PCR amplification reaction conditions were: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C 3 Minutes, last 72 ° C for 5 minutes.

 [0056] 1. Preparation of F180A mutant

 [0057] The following primer pairs were used for F180A-F: 5'

 GTTCAAACTGCACGACGCGGGTGCTCGTGGTGTTTC 3' and F180A-R: 5'

 GAAACACCACGAGCACCCGCGTCGTGCAGTTTGAAC 3', using the plasmid pRSET-nampt constructed in the first part of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of the F180A mutant gene, electrophoresed by 1% agarose gel The amplified product was isolated and recovered using a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-F180A. The plasmid pRSET-F180A was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-F180A was extracted, and the point mutation introduced was confirmed by DN A sequencing. The amino acid sequence of the F180A mutant is mutated from Phe (F) to Ala (A) at the 180th position, and the amino acid sequence thereof is as shown in SEQ ID NO: 3, as compared with the parent amino acid sequence shown in SEQ ID NO: 2. .

 2. Preparation of F180W mutant

 [0059] The following primer pair F180W-F: 5'

 GTTCAAACTGCACGACTGGGGTGCTCGTGGTGTTTC 3' and F180W-R: 5' GAAACACCACGAGCACCCCAGTCGTGCAGTTTGAAC 3', using the plasmid pRSET-nampt constructed in the first part of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of F180W mutation The gene was separated by electrophoresis on a 1% agarose gel and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-F180W. The plasmid pRSET-F180W was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-F180W was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the F180W mutant was mutated from Phe (F) to Trp (w) at the 180th position as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.

 [0060] 3. Preparation of A182Y mutant

[0061] The following primer pair A182Y-F: 5' The plasmid pRSET-nampt constructed in the first part was used as a template, and the A182Y mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions, separated by 1% agarose gel electrophoresis and recovered by commercial kit. The product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain the plasmid pRSET-A182Y. The plasmid pRSET-A182Y was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DN A of the plasmid pRSET-A182Y was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the A182Y mutant was mutated from Ala (A) to Tyr (Y) at position 182 compared to the parent amino acid sequence set forth in SEQ ID NO: 2.

4. Preparation of E231A mutant

 [0063] The following objects are used: Ε231 A-F: 5' CTATCCCGGCTATGGCGCACTCTACCGTTAC

3' and Ε231 AR: 5' GTAACGGTAGAGTGCGCCATAGCCGGGATAG 3', using the plasmid pRSET-nampt constructed in the first part of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of the E231A mutant gene The amplified product was separated by electrophoresis on a 1% agarose gel and recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-E231A. The plasmid pRSET-E231A was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DN A of the plasmid pRSET-E231A was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the E231A mutant was mutated from Glu (E) to Ala (A) at position 231 compared to the parent amino acid sequence as shown in SEQ ID NO: 2.

[0064] 5. Preparation of E231Q mutant

Using the following pair of E231Q-F: 5' CTCTATCCCGGCTATGCAGCACTCTACCGTTACC 3' and E231Q-R: 5' GGTAACGGTAGAGTGCTGCATAGCCGGGATAGAG 3', using the plasmid pRSET-nampt constructed in the first part of Example 6 as a template, using the above PCR amplification reaction System and P CR amplification reaction conditions for high-fidelity PCR amplification of E231Q mutant gene, electrophoresis by 1% agarose gel The amplified product was recovered by using a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-E231Q. The plasmid pRSET-E231Q was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-E231Q was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the E231Q mutant was mutated from Glu (E) to Gin (Q) at position 231 as compared to the parent amino acid sequence set forth in SEQ ID NO: 2.

 [0066] 6. Preparation of D298A mutant

 [0067] The following pairs of objects D298A-F: 5' TATCCGTCCGGCGTCTGGTGACCC 3' and D298A-R:

 5' GGGTCACCAGACGCCGGACGGATA

 3', using the plasmid pRSET-nampt constructed in the first part of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of D298A mutant gene, separated by 1% agarose gel electrophoresis The amplified product is recovered by a commercial kit, and the amplified product is ligated with the vector pRSET-A.

(Specific reference to the first part of Example 6), the plasmid pRSET-D298A was obtained. The plasmid pRSET-D298A was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-D298A was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the D298A mutant was changed from Asp (D) to Ala (A) at the 298th position as compared with the parent amino acid sequence as shown in SEQ ID NO: 2.

7. Preparation of D298N mutant

The PCR amplification reaction was carried out using the plasmid pRSET-nampt constructed in the first part of Example 6 using the following plasmid pair D298N-F: 5' GTTGTTATCCGTCCGAATTCTGGTGACCCGCCG 3' and D298N-R: 5' CGGCGGGTCACCAGAATTCGGACGGATAACAAC 3' System and PC R amplification reaction conditions High-fidelity PCR amplification of the D298N mutant gene, separation by 1% agarose gel electrophoresis and recovery of the amplified product with a commercial kit, and then the amplification product was ligated with the vector pRSET-A (specific reference) Example 6 Part I), plasmid pRSET-D298N was obtained. The plasmid pRSET-D298N was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. Extract the plasmid pRSET-D298N from D NA, the point mutation introduced by DNA sequencing was confirmed to be correct. The amino acid sequence of the D298N mutant was mutated from Asp (D) to Asn at position 298 compared to the parent amino acid sequence set forth in SEQ ID NO: 2.

N).

 8. Preparation of D298E mutant

 [0071] The following primer pair D298E-F: 5'

 GTTGTTATCCGTCCGGAATCTGGTGACCCGCCGTTC 3' and D298E-R: 5' GAACGGCGGGTCACCAGATTCCGGACGGATAACAAC 3', using the plasmid pRSET-nampt constructed in the first step of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification The D298E mutant gene was isolated by electrophoresis on a 1% agarose gel and the amplified product was recovered using a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-D298E. The plasmid pRSET-D298E was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-D298E was extracted, and the point mutation introduced was confirmed by DN A sequencing. The amino acid sequence of the D298E mutant was mutated from Asp (D) to Glu (E) at position 298 as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.

 9. Preparation of D338N mutant

 [0073] The following primer pair D338N-F: 5'

GAGTCAGCGTTAACACCATTACCCTGGATAACACGAAC 3', using the plasmid pRSET-nampt constructed in the first part of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of D338N mutant gene, electrophoresed by 1% agarose gel The amplified product was isolated and recovered using a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain the plasmid pRSET-D338N. The plasmid pRSET-D338N was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-D338N was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the D338N mutant was mutated from Asp (D) to Asn (N) at position 338 as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.

10. Preparation of D338E Mutant [0075] With the following pair of objects D338E-F: 5' GTTATCCAGGGTGAAGGTGTTAACGCTGAC

3' and D338E-R: 5' GTCAGCGTTAACACCTTCACCCTGGATAAC 3', using the plasmid pRSET-nampt constructed in the first part of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of D338E mutant The gene was isolated by electrophoresis on a 1% agarose gel and the amplified product was recovered using a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-D338E. The plasmid pRSET-D338E was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-D338E was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the D338E mutant was mutated from Asp (D) to Glu (E) at position 338 as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.

 11. Preparation of D377A mutant

 [0077] With the following pair of objects D377A-F: 5' CACCCGCACCGTGCGACCCAGAAATTC

 3' and D377A-R: 5' GAATTTCTGGGTCGCACGGTGCGGGTG 3', using the plasmid pRSET-nampt constructed in the first step of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of D377A The mutant gene was separated by electrophoresis on a 1% agarose gel and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-D377A. The plasmid pRSET-D377A was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-D377A was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the D377A mutant was mutated from Asp (D) to Ala (A) at position 377 as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.

 12. Preparation of D377N mutant

 [0079] The following primer pair D377N-F: 5'

GAGCGAATTTCTGGGTATTACGGTGCGGGTGTTGC 3', using the plasmid pRSET-nampt constructed in the first porphin of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of the D377N mutant gene, 1% agarose The gel electrophoresis was separated and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (refer to the first embodiment in detail). Part), the plasmid pRSET-D377N was obtained. The plasmid pRSET-D377N was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-D377N was extracted, and the point mutation introduced was confirmed by DN A sequencing. The amino acid sequence of the D377N mutant was mutated from Asp (D) to Asn (N) at position 377 as compared to the parent amino acid sequence set forth in SEQ ID NO: 2.

13. Preparation of D377E mutant

 [0081] The following primer pairs were used for D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG

 3' and D377E-R: 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', using the plasmid pRSET-nampt constructed in the first part of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of D377E mutant The gene was isolated by electrophoresis on a 1% agarose gel and the amplified product was recovered using a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-D377E. The plasmid pRSET-D377E was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-D377E was extracted, and the point mutation introduced was confirmed by DN A sequencing. The amino acid sequence of the D377E mutant was mutated from Asp (D) to Glu (E) at position 377 as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.

 14. Preparation of E231Q/D338E Mutant

 [0083] The following primer pairs were used for D338E-F: 5' GTTATCCAGGGTGAAGGTGTTAACGCTGAC

3' and D338E-R: 5' GTCAGCGTTAACACCTTCACCCTGGATAAC 3', using the plasmid pRSET-E231Q constructed in the fifth subsection of Example 6, Part 2, as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high fidelity PCR The E231Q/D338E mutant gene was amplified, separated by 1% agarose gel electrophoresis and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET. -twenty one. The plasmid pRSET-21 was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-2 1 was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the E231Q/D338E mutant was mutated from Glu (E) to Gin (Q) at position 231 and by Asp (D) at position 338, compared to the parent amino acid sequence set forth in SEQ ID NO: 2. ) Mutation to Glu (E). 15. Preparation of E231Q/D377E Mutant

 [0085] The following primer pairs were used for D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG

 3' and D377E-R: 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', using the plasmid pRSET-E231Q constructed in the fifth subsection of the second part of Example 6 as a template, using the above PCR amplification reaction system and PC R amplification reaction conditions for high fidelity The E231Q/D377E mutant gene was amplified by PCR, separated by 1% agarose gel electrophoresis and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (refer to the first part of Example 6 for specific reference) to obtain a plasmid. pRSET-22. The plasmid pRSET-22 was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-22 was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the E231Q/D377E mutant was mutated from Glu (E) to Gin (Q) at position 231 and by Asp (D) at position 377, compared to the parent amino acid sequence as shown in SEQ ID NO: 2. ) Mutation to Glu (E).

[0086] 16. Preparation of D338E/D377E mutant

 [0087] The following primer pairs were used for D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG

 3' and D377E-R: 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', using the plasmid pRSET-D338E constructed in the 10th subsection of the second part of Example 6 as a template, using the above PCR amplification reaction system and P CR amplification reaction conditions for high fidelity The D338E/D377E mutant gene was amplified by PCR, separated by 1% agarose gel electrophoresis and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (refer to the first part of Example 6 for specific reference) to obtain a plasmid. pRSET-23. The plasmid pRSET-23 was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DN A of the plasmid pRSET-23 was extracted, and the point mutation introduced was confirmed by DNA sequencing. Compared with the parent amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the D338E/D377E mutant was mutated from Asp (D) to G1 u (E) at position 338 and by Asp at position 377 ( D) Mutation to Glu (E).

[0088] 17. Preparation of E231Q/D338E/D377E Mutant

 [0089] The following primer pairs were used for D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG

3' and D377E-R: 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', the plasmid pRSET-21 constructed in the 14th subsection of the second part of Example 6 as a template, the above PCR amplification reaction system and PCR Amplification reaction conditions were carried out by high-fidelity PCR amplification of the E231Q/D338E/D377E mutant gene, which was separated by 1% agarose gel electrophoresis and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference) Example 6 Part I), plasmid pRSET-31 was obtained. The plasmid pRSET-31 was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-31 was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the E231Q/D338E/D377E mutant was mutated from Glu (E ) to Gin (Q) at position 231 and from Asp at position 338, compared to the parent amino acid sequence as shown in SEQ ID NO: 2. (D) Mutation to Glu (E), mutation from Asp (D) to Glu (E) at position 377.

[0090] 18. Preparation of E231Q/D298A/D338E/D377E mutant

 Using the following primer pair D298A-F: 5' TATCCGTCCGGCGTCTGGTGACCC 3' and D298A-R: 5' GGGTCACCAGACGCCGGACGGATA 3', the plasmid pRSET-31 constructed in the 17th subsection of the second part of Example 6 was used as a template, using the above PCR The amplification reaction system and the PCR amplification reaction conditions were subjected to high-fidelity PCR amplification of the E231Q/D298A/D338E/D377E mutant gene, which was separated by 1% agarose gel electrophoresis and the amplified product was recovered by a commercial kit, and then the amplification product was used. The vector pRSET-A was ligated (specifically with reference to the first part of Example 6) to obtain plasmid pRSET-41. The plasmid pRSET-41 was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. The DNA of the plasmid pRSET-41 was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the E231Q/D298A/D338E/D377E mutant was mutated from Glu (E ) to Gin (Q) at position 231, at position 298, compared to the parent amino acid sequence set forth in SEQ ID NO: 2. It was mutated from Asp (D) to Ala (A), from Asp (D) to Glu (E) at position 338, and from Asp (D) to Glu (E) at position 377.

 [0092] Third, the extraction of enzymes

The plasmid pRSET-nampt containing the parent nicotinamide phosphoribosyltransferase gene and the plasmid pRSET-F180A, pRSET-F180W, pRSET-A182Y, pR SET-E231A, pRSET- containing the nicotinamide phosphoribosyltransferase mutant gene. E231Q^ pRSET-D298A, pRSET-D298N, pRSET-D298E, pRSET-D338N, pRSET-D338E, pRSET-D377A, pRSET-D377N, pRSET-D377E, pRSET-21, pRSET-22, pRSET-23, pRSET-31 and pRSET-41 transforms competent cells The bacterial cell E. coli BL21 was cultured on a Luria broth (LB) plate (containing 50 mg/L kanamycin) for 24 hours at 37 °C. Inoculate a single clone in 50 ml of LB liquid medium (including 50

 In mg/L kanamycin), culture at 30 ° C for 16-20 hours. The cells were collected by centrifugation, and the equivalent amount of bacteria was weighed and suspended in a ratio of 1:4 (pH).

 7.5) Medium. The bacterial cells are then lysed with ultrasound. Centrifuge (4-10 ° C, 12000 rpm, 10 minutes) and collect the supernatant to obtain the parent nicotinamide phosphoribosyltransferase and a series of supernatant proteins of nicotinamide phosphoribosyltransferase mutants, which can be used for enzyme activity. Determination of the biocatalytic preparation of nicotinamide mononucleotides.

[0094] Determination of enzyme activity

[0095] Formulation of substrate solution: containing 60 mM nicotinamide, 25 mM PRPP, 18 mM MgCl ₂

 , 15 mM KC1 and 100 mM Tris buffer buffer, adjusted to pH 7.5. Take 900 μl of each substrate solution, respectively, and then add 100 μl of the same concentration of the parent nicotinamide phosphoribosyltransferase obtained in the third part of Example 6 and the supernatant protein of each nicotinamide phosphoribosyltransferase mutant. The solution was reacted at 37 ° C for 10 minutes, and 10 (VL25 < 3⁄4 trichloroacetic acid was added to terminate the reaction. The content of nicotinamide mononucleotide (NMN) in the reaction solution was determined by high performance liquid chromatography (HPL C), and The specific enzyme activity of each enzyme was calculated, and the specific activity of the parent nicotinamide phosphoribosyltransferase was referred to as reference 100. The relative activities of the parent and each mutant are shown in Table 1.

Table 1 Enzyme activity of nicotinamide phosphoribosyltransferase

[Table 1]

 [0097] V. Preparation of Nicotinamide Mononucleotide

[0098] A substrate solution was added to the reaction vessel containing 75 mM nicotinamide, 75 mM disodium pyrophosphate, 50 mM AMP, 15 mM MgCl ₂ 10 mM KC1 and 100 mM Tris-HCl buffer were adjusted to pH 7.0-7.5. Then add the following catalytic enzyme: the supernatant protein solution of the nicotinamide phosphoribosyltransferase mutant (F180A) obtained in the third part of Example 6 10 ml / L substrate solution, adenine phosphoribosyltransferase 20 g / L substrate solution . After stirring uniformly, the reaction was carried out, stirring was continued during the reaction (stirring speed 50 rpm), the reaction temperature was controlled at 37 ° C, and the pH was maintained at 7.0-8.0. After 5 hours of reaction, a crude nicotinamide mononucleotide solution (containing NMN49.6 mM) was obtained.

), after filtration, purification, and drying, the finished nicotinamide single nucleotide is obtained.

Claims

Claim  [Claim 1] A method for producing a nicotinamide mononucleotide, which comprises: using nicotinamide, pyrophosphoric acid or a salt thereof and AMP as a raw material, in nicotinamide phosphoribosyltransferase and adenine phosphoribosyltransferase The reaction takes place under catalysis to produce a nicotinamide mononucleotide. [Claim 2] The method for producing a nicotinamide mononucleotide according to claim 1, wherein the reaction is carried out at a temperature of 30 to 50 ° C and a pH of 6.5 to 8.5. [Claim 3] The method for producing a nicotinamide mononucleotide according to claim 1, wherein the reaction is carried out in the presence of Mg ²⁺ and K + . [Claim 4] The method for producing a nicotinamide mononucleotide according to claim 1, wherein the reaction is carried out in a Tris-HCl buffer. [Claim 5] The method for producing a nicotinamide mononucleotide according to claim 1, wherein: the concentration of the nicotinamide is 1 to 150 mM, and the concentration of the pyrophosphoric acid or a salt thereof is 1 to 100 mM The concentration of the AMP is 1-100 mM. [Claim 6] The method for preparing a nicotinamide mononucleotide according to claim 1, wherein the molar ratio of nicotinamide, pyrophosphoric acid or a salt thereof, and AMP in the raw material is 1-4:1 2:1. [Claim 7] The method for producing a nicotinamide mononucleotide according to claim 6, wherein a molar ratio of nicotinamide, pyrophosphoric acid or a salt thereof, and AMP in the raw material is 2:1.5:1. [Claim 8] The method for producing a nicotinamide mononucleotide according to any one of claims 1 to 7, wherein the nicotinamide phosphoribosyltransferase is a protein of the following: or (b): (a) a protein having the amino acid sequence of SEQ ID NO: 3,  (b) Substituting, deleting or adding one or several amino acids in the (a) defined amino acid sequence and having a specific amino acid sequence such as SEQ ID NO with nicotinamide and PRPP as substrates : 2 The parental high nicotinamide phosphoribosyltransferase catalytically active protein derived from (a). [Claim 9] The method for producing a nicotinamide mononucleotide according to claim 8, wherein the nicotinamide phosphoribosyltransferase is selected from the amino acid sequence shown in SEQ ID NO: At least one of the following sites has at least one mutation: 180th, 182th, 231rd, 298th, 338th, and 377th. [Claim 10] The method for producing a nicotinamide mononucleotide according to claim 9, wherein the nicotinamide phosphoribosyltransferase has at least one of the following mutations: F180A, F180W, Al 82Y, Ε231Α, E231Q , D298A, D298N, D298E, D338N, D338E, D377A, D377N and D377E.